Method and apparatus for cell recovery

ABSTRACT

A method and apparatus for recovering cells from stool are provided for diagnosing colorectal cancer from stool naturally voided by multiple specimens. The method includes the steps of preparing a sample of naturally voided and collected stool, to which sample a buffer solution is added, causing cancer cells in the sample from which the impurities have been removed to be adsorbed on a solid carrier, and recovering the cancer cells thus adsorbed.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of application Ser. No.10/773,316, filed Feb. 9, 2004, the disclosure of which is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of recovering cells fromstool.

2. Background Art

In Europe and the United Sates, colorectal cancer is a leading cause ofcancer deaths. In Japan too, the number of patients of colorectal canceris increasing sharply in recent years. This is believed due to the moreWesternized, meat-oriented diet adopted by the Japanese. About 60thousand people in Japan are diagnosed with colorectal cancer everyyear. On an organ-by-organ basis, the number of deaths from colorectalcancer is third highest, following stomach and lung cancer, and thenumber is expected to increase. However, colorectal cancer is known tobe almost 100% curable by operation if detected early. Thus, colorectalcancer is included in an early cancer screening scheme, and manyexamination methods have been developed.

Examples of the examination methods for the early detection ofcolorectal cancer include barium enema and colonoscopy. In an enemaexamination, barium is injected into the large intestine and allowed toattach to the mucous membrane surface of the intestine, so that thesurface irregularities can be examined by X-ray. The colonoscopyinvolves the direct observation of the inside of the large intestine byan endoscope. These methods have high sensitivity and specificity to thedetection of colorectal cancer. In addition, the colonoscopy has theadvantage that early cancer or precancerous polyps can be removed.However, these examination methods put a large burden on the patientsand are costly. Particularly, the colonoscopy requires skills inoperation and is associated with the risk of complications such asbleeding or perforation. Accordingly, they are not suitable forscreening the general public with no subjective symptoms for colorectalcancer.

As a method for the primary screening of the general public forcolorectal cancer, a fecal occult blood test is widely used. In thefecal occult blood test, the presence or absence of bleeding in thebowel is detected by examining the presence of hemoglobin contained inthe stool, in order to indirectly predict the development of colorectalcancer.

The fecal occult blood test can be roughly divided into two types,namely a chemical examination method and an immunological examinationmethod. The chemical fecal occult blood test takes advantage of theperoxidase activity of hemoglobin, and it utilizes the reaction in whichguaiac contained in a filter paper is turned into a blue-green oxide dueto the active oxygen produced upon breakdown of hydrogen peroxide thatis added as a matrix. Commercially available filter papers for such anexamination include Hemoccult II filter paper (Fujisawa PharmaceuticalCo., Ltd.) and Shionogi B filter paper (Shionogi & Co., Ltd.).

The immunological fecal occult blood test utilizes the specific bindingof antihuman hemoglobin antibody to human hemoglobin. The method is wellon its way to becoming the dominant fecal occult blood test method dueto its high specificity. Examples include a reversed-passivehemagglutination method (Immudia Hem-Sp, Fujirebio Inc.), amagnetic-particle agglutination and gradient method (Magstream Hem-Sp,Fujirebio Inc.), and a latex agglutination method (Immunoccult, ChugaiPharmaceutical Co., Ltd.).

Though the fecal occult blood test is widely used for the screening ofcolorectal cancer, some people are voicing suspicion over the efficacyof the test. A positive result in a chemical fecal occult blood testusing the Hemoccult II requires 20 mg per day of bleeding in the largeintestine. However, in an actual colorectal cancer patient, the amountof bleeding is thought to be 10 mg or less. As a result, the sensitivityof the fecal occult blood test is approximately 26%, and there have beenreports that only about one quarter of the actual colorectal cancerpatients can be detected and the remaining three quarters are overlooked(Jama, Vol. 269, 1262-7, 1993). Further, only 8.3% of all the positivesactually had colorectal cancer and many false positives were included.

Thus, there is a need for the development of a new primary screeningtest method with better accuracy. As a possible candidate for thatpurpose, a testing method utilizing cancer cells shed onto the stool isgaining attention. Compared with the fecal occult blood test thatdetects the bleeding in the bowel which occurs indirectly in associationwith a colorectal cancer, the method of the present invention directlyexamines cancer cells and can therefore provide a more reliable testingmethod.

As a method of examining cancer cells in the stool, JP PatentPublication (Kohyo) No. 2002-515973 A (WO97/28450), for example,describes a method for genetic diagnosis utilizing nucleic acidsdirectly extracted from stool. Concrete examples of the genetic mutationdetecting method include the sequence method, the PCR-RFLP (polymerasechain reaction-restriction enzyme fragment length polymorphism) method,the SSCP (single-stranded conformational polymorphism) method, and thePTT (protein truncation test) method. Apart from the detection ofgenetic mutations, diagnostic methods that utilize the instability of amicrosatellite (MSI, microsatellite instability) or the appearance of along DNA (L-DNA) as indicators are known.

As the genes that can be examined for genetic mutations, K-ras, APC, P53and DCC, for example, are widely known. Searches actively continue evennow for genes that can be new objects of examination, taking advantageof different expression levels and utilizing a microarray, for example.A method has also been proposed that uses the expression pattern of asplicing variant of the CD44 gene as a marker.

One problem associated with these testing methods is the fact that thenucleic acids in the stool derive from various bacteria and normalcells, and that the ratio of genes deriving from cancer cells collectedfrom the stool is very small (about 0.05%). This poses a significanthindrance in the examination of mutations in cancerous cell-derivedgenes or subtle changes in expression patterns, thus making thepractical application of the method difficult.

Thus, methods of collecting cancer cells directly from stool andexamining them have been considered, with a view to providing a morereliable colorectal cancer diagnosis. In order to collect cancer cellsfrom stool, two steps are important. One is the step of exfoliatingcells from the stool, and the other is the step of collecting theexfoliated cells.

JP Patent Publication (Kohyo) No. 11-511982 A (1999) (WO97/09600)reports a preliminary processing method whereby the stool is cooled inthe step of exfoliating cells therefrom, and then the cells existingunder the surface of the stool are exfoliated. Specifically, claim 1 ofthe document recites the step of “cooling the stool to a temperaturebelow its gel freezing point,” and in this method, the surface of thestool cooled and frozen is scraped and the cancer cells existing in thesurface are exfoliated. Methods have also been reported that employ adevice called stomacher that is capable of mildly pulverizing a solidmatter, wherein the entire stool is suspended and cells are exfoliated.

In the step of recovering the exfoliated cells, a centrifugal separationmethod utilizing a Percoll (Int J Cancer, Vol. 52, 347-50, 1992) or arecovery method utilizing magnetic beads to which an antihuman antibodyis bound (Lancet, Vol. 359, 1917-9, 2002, Apmis, Vol. 110, 239-46, 2002)have been reported. Particularly, magnetic beads to which Ber-EP4antibody is bound that specifically binds to epithelium cells arecommercially available (Dynabeads Epithelial Enrich, Dynal Biotech), andthey are known to bind to colorectal cancer cell lines. In PatentDocument 2 too, the Ber-EP4 binding magnetic beads are utilized forrecovering cancer cells from stool.

SUMMARY OF THE INVENTION

In a large-scale colorectal cancer screening performed on the generalpublic, a large amount of multiple specimens must be processed by anautomated system. The method in which cells are directly recovered fromstool and examined for colorectal cancer is excellent in reliability.However, in the existing method whereby cells are recovered from thesurface of stool that is frozen by cooling, as described in JP PatentPublication (Kohyo) No. 11-511982 A (1999) (WO97/09600) (to be hereafterreferred to as a cooling method), the operation is bothersome and thescreening cannot be performed on a large scale.

In order to realize the automated processing of a large amount ofmultiple specimens, the operation must be simplified and the time ofoperation must be reduced. In the cooling method, there is the step ofcentrifugation, which takes time and does not render itself toautomation and thus makes the processing of multiple specimensdifficult. Further, the specimen cooling operation requires large-sizedequipment and makes the stool processing operation complicated.

For the determination of colorectal cancer using the cells recoveredfrom stool, a method employing a cytological analysis for identifyingcolorectal cancer is very effective. However, in the conventionalcooling method, the cells are damaged by the cooling operation, therebymaking it difficult to perform a cytological analysis.

Further, in the cooling method, the cells below the surface of the stoolare exfoliated and recovered. In the ascending colon portion near thesmall intestine, the stool is in the form of muddy water, and it isbelieved that the most cancer cells exfoliated from the walls of thelarge intestine are later taken into the inner core of the stool duringthe process of forming a solid feces. Thus, it is likely that cancercells deriving from the ascending colon cannot be recovered by thecooling method.

To solve the aforementioned problems, the method and apparatus for therecovery of cancer cells according to the invention are operated at roomtemperature.

Specifically, in contrast to the cooling method in which the surface ofa cooled and frozen stool is scraped and then cancer cells existing inthe surface of the stool are exfoliated, the method of recovering cellsaccording to the invention includes the steps of, at room temperature,preparing a sample to which a buffer solution is added, causing a cancercell in the sample from which the impurity has been removed to beadsorbed on a solid carrier, and recovering the thus adsorbed cancercell. Thus, all of the steps for the recovery of cancer cells can beconducted without temperature control. Similarly, the apparatus forrecovering cells according to the invention includes a bag for storing asample comprising a buffer solution and stool at room temperature, and acontainer in which a solid carrier for the adsorption of a cell in thesample is stored. Thus, the cell recovery apparatus does not require atemperature control means. As a result, the cell recovering method andapparatus according to the invention can simplify the cell recoveryoperation and allow cancer cells in stool to be recovered stably andefficiently, thereby providing a high determination accuracy.

FIG. 1 shows the temperature dependency of the cell stability in stooland the antigen-antibody binding reaction rate. As shown in FIG. 1, whenthe temperature is low, the cell stability in stool decreases and,particularly, in a frozen state at temperatures of 4° C. or lower, thecancer cells could be destroyed, which would make it difficult to carryout the subsequent cytological analysis. On the other hand, with regardto the antigen-antibody binding reaction rate, the antibody isdeactivated as the temperature increases, thus also making it difficultto carry out the subsequent immunological operations. Thus, inaccordance with the invention, a range of temperatures around roomtemperature, or a temperature range between 5° C. and 40° C., orpreferably between 15° C. and 35° C., is adopted so as to make both thecell stability and the antigen-antibody binding reaction rate compatiblewith each other. All of the steps from the recovery of cancer cells tothe adsorption may be conducted at room temperature.

Further, the invention provides a novel filter system capable ofhandling a suspension of stool as a whole. A funnel-shaped filterimproves the efficiency of filtering of a stool suspension, reduces thetime required for operation, and eliminates the operation ofcentrifugation, thereby greatly simplifying the entire operation.Further, as cells are recovered from the stool as a whole including thecentral portion thereof, cancer diagnosis can be conducted on the entirelarge intestine. On the other hand, a multi-stage filtering apparatusallows the cells to be trapped on a film and thus condensed. The film inwhich the cells are trapped can then be recovered, so that the inventioncan be adapted for an automated system.

Further, conditions for a protocol have been analyzed, including theaddition of blood serum into a stool suspension, and a simplifiedprotocol has been developed whereby all of the steps can be operated atroom temperature.

In accordance with the method and apparatus of the invention for cellrecovery, good, living cancer cells can be recovered from stool at roomtemperature, in contrast to the cooling method as disclosed in JP PatentPublication (Kohyo) No. 11-511982 A (1999) (WO97/09600), whereby thesurface of a cooled and frozen stool is scraped and then cancer cellsexisting in the surface of the stool are exfoliated. Thus, in accordancewith the invention, the recovered cells can be subjected to cytological,immunological and biochemical analyses with high accuracy. Also, themethod and apparatus of the invention for recovering cells can utilize acell deriving from early colorectal cancer or the stool as a whole as aspecimen, cancer cells deriving from the ascending colon, which aredifficult to detect endoscopically, can be recovered. Thus, theinvention can provide a highly reliable examination method. Further, themethod and apparatus of the invention have eliminated the centrifugationand cooling operations so that the operation can be simplified andperformed in less time. Thus, an automated total system for colorectalcancer examination can be constructed using the method and apparatus ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph indicating the temperature dependency of the cellstability in stool and the antigen-antibody binding reaction rate.

FIG. 2 schematically shows a standard protocol for a method ofrecovering colorectal cancer cell from stool.

FIG. 3 shows the shape of a funnel-shaped filter for filtering a stoolsuspension.

FIG. 4 shows the concept of a total system for colorectal cancerexamination according to the present invention.

FIG. 5 shows the results of analysis of conditions for the cell recoverymethod. FIG. 5(A) shows the relationship between the temperature atwhich the cells and the magnetic beads bind to one another, and the cellrecovery rate. FIG. 5(B) shows the relationship between the presence orabsence of blood serum in the medium and the cell recovery rate.

FIG. 6 compares the cell recovery rate according to the conventionalmethod and that according to the magnetic bead method of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

(Recovery of Cells from Stool)

FIG. 2 shows the standard protocol for the method of recovering cellsfrom stool according to the present invention. Hereafter, the procedureof the standard protocol will be described.

(Step 1: Recovery of Specimens)

The stool used in the present invention is stool naturally voided by ahuman. A solid stool is used and diarrheal stool is not used. Neither isthat stool used that has been voided after the subject had taken acompulsive relieving agent such as a laxative or barium for intestinalexamination. There is no need for the subject to exercise any particulardiet control prior to the test.

The stool for specimen is recovered on a dish- or sheet-shapeddisposable container, and an appropriate amount is put into a stomacherbag. Other methods of stool recovery may be employed, such as by meansof a stick-type stool recovery apparatus or a stamp-type recoveryapparatus, as long as the method is capable of recovering an appropriateamount of stool. As the stomacher bag, a commercially availablefilterless bag is employed. A stomacher bag with a filter may be used.The stomacher herein refers to mixers in general for the pulverizationof specimens contained in a bag-like container. The stomacher bag refersto a commercially available bag specifically designed for stomachers.Other substitutes may be used as long as they can be used withstomachers.

The stool recovered from the subject should preferably be used withinthree hours but may be used within up to approximately ten hours. Thestool can be stored at room temperature during that period and there isno need to store it in a refrigerated or frozen state.

The amount of stool used is preferably in the range between about 5 gand 80 g. However, the amount may range from about 0.5 g to 200 g.

A suspension medium is put into the stomacher bag in which the stool isrecovered. The medium is a Hanks solution. However, any conventionalmedium that is used in experiments involving cells may be used. Specificexamples include PBS, PBS (−) and media for the cultivation of variouscells (MEM, DMEM, RPMI).

The amount of medium added may be varied depending on the amount orstate of stool. However, the amount is preferably 1 mL or more per 1 gof stool. By adding 200 mL of medium per one stomacher bag, any of theaforementioned amounts of stool may be accommodated.

Blood serum is added to the medium. The concentration of blood serum ispreferably 10% but may be in the range between 0.5% and 20%. The bloodserum is preferably FBS (fetal bovine serum) but may be CS (calf serum).

The stomacher bag containing the stool with medium is sealed with asealer. Leakage of the suspension can be more completely prevented bycovering the stomacher bag with another stomacher bag. The thus sealedstomacher bag is processed by a stomacher to produce a stool suspension.This step of recovering a specimen is conducted at room temperature.However, if it takes time between the recovery of specimen andfiltration, the stool suspension may be stored in a cooler box, forexample.

(Step 2: Filtration)

The suspension is filtered by a filter in a draft to remove residualmatter. When a stomacher bag with a filter is used, the suspension isfiltered by the filter in the bag and the filtered solution isrecovered.

When a stomacher bag with no filter is used, a new filtering apparatusis used for filtering the suspension.

The filtering apparatus is used either with a single filter or in amultiple-stage arrangement made up of filters with various sizes. When asingle filter is used, the size is preferably about 500 μm. However, thesize may be in the range between 40 to 1500 μm, or preferably between400 and 1000 μm. When the multiple-stage arrangement is used, thesuspension is caused to flow from a filter with a larger size to onewith a smaller size. The size of the filters for the multiple-stagefiltering may be between about 40 to 2000 μm. By disposing a filter witha size of 10 μm or less in the final stage, the cells can be captured onthe final filter.

The filtering apparatus may be either of a free-fall type or a suctionfiltering type.

FIG. 3 shows an example of the shape of filter. The funnel shape of thefilter has dimensions such that the diameter of the opening is 60 mm,the diameter of the bottom is 20 mm, the height is 200 mm, and theheight when the filter is inserted into a container is 170 mm, forexample. The filter shown in FIG. 3 is a funnel-type three-dimensionalfilter having filtering sides. Preferably, a bottom-surface type filterhaving a filtering surface only at the bottom may be used. Further, thesurface of the filter may be provided with fold-like irregularities suchthat the area of contact with the suspension can be increased.

The material of the filter is preferably nylon. Other materials may beused as long as they are capable of allowing a filter with a desiredsize or shape to be produced. Specific examples include polyester,polyethylene, and polypropylene. This filtering step is conducted atroom temperature.

(Step 3: Magnetic Beads Reaction)

The cells contained in the filtered solution are recovered using acarrier having an affinity for cancer cells. The carrier is made ofmagnetic beads having bound to the surfaces thereof an antibody with anaffinity for cancer cells. Specifically, Ber-EP4 antibody-bindingmagnetic beads commercially available from Dynal Biotech (DynabeadsEpithelial Enrich) are used. Besides Ber-EP4, other antibodies having anaffinity for colorectal cancer cells may be used. Besides antibodies,aptamars or ligands having an affinity for colorectal cancer cells maybe used.

Forty mircrolitters of magnetic beads are added per tube containingabout 20 to 45 mL of dispensed filtered solution. The amount of magneticbeads may be varied between about 20 μL and 400 μL.

The filtered solution to which the magnetic beads have been added isblended using a mix rotor, such that the cells in the filtered solutionare bound to the magnetic beads. The blending is preferably conducted atroom temperature or in a cold room at 4° C., preferably for 10 minutesor more. This step of reacting magnetic beads is carried out at roomtemperature.

(Step 4: Magnetic Separation)

The tubes containing the blended filtered solution are placed on amagnetic stand and is then shook for 15 minutes, such that the magneticbeads are collected on the side of the tube. The shaking is preferablyconducted for 10 minutes or more. The shaking may be conducted in anymanner, such as a see-saw motion, rotation or gyration, as long as thefiltered solution can be gradually blended.

After the magnetic beads have attached to the wall surface, the filteredsolution is removed. After the removal of solution, the tubes aredetached from the magnetic stand and washed with the aforementionedmedium, and then the beads washing solution is recovered. The amount ofmedium is 500 μL per tube but may be varied as desired in light of thesubsequent experiment. This step of magnetic separation is conducted atroom temperature.

(Step 5: Magnetic separation, Eppendorf Tube)

The washing solution is recovered into an Eppendorf tube or the likesmaller than the previously used tubes. The tube containing the washingsolution is immediately attached to a dedicated magnetic stand. Afterthe magnetic beads have been collected on the side walls of theEppendorf tube, the supernatant is removed to obtain pellets ofcell-bead complexes. This step of magnetic separation and Eppendorf tubeis conducted at room temperature.

(Diagnosis for Colorectal Cancer)

The pellets recovered by the present standard protocol are then used asspecimens for the determination of colorectal cancer. The determinationof cancer is based either on cells themselves or a substance extractedfrom cells. When cells themselves are used, the pellets are usedimmediately after recovery. When an extracted substance is used, thepellets can be stored in a frozen state at −80° C.

When cells themselves are used, the cells are stained with Papanicolaoustain and then observed by a microscope for determination. If the ratioof nucleus to cytoplasm (N/C) is high and atypical cells with chromatincondensation are identified, the cells are determined to be cancerous.Other staining methods may be used as long as they are capable ofidentifying cancer cells. Besides conventional staining, immunostainingthat utilizes a cancer-cell specific antibody may be used.

It is possible to extract DNA or RNA from cells and utilize them forcancer determination. For the extraction of DNA or RNA, nuclear acidextraction kits available from various companies can be employed.Specific examples of such kits include Dynabeads DNA DIREIC Universalfrom Dynal Biotech, QIAamp DNA MiniKit from Qiagen, and SepaGene fromSanko Junyaku Co., Ltd. for DNA extraction. For the extraction of RNA,ISOGEN from Nippon Gene Co., Ltd. and TRIzol Reagent from Invitrogen canbe cited. The extracted nucleic acids can be utilized for the variousmethods mentioned in the description of the related art.

(Stool Processing Total System)

The concept of a stool processing total system according to the presentinvention is shown in FIG. 4. Collected specimens are suspended using astomacher. For the filtering of the suspension, an apparatus in whichsingle funnel-shaped filters are arranged as shown in FIG. 4 can beused. The apparatus may be replaced with a multi-stage filteringapparatus. The filtering apparatus is equipped with a suction filteringfunction. The filtered liquid is dispensed and agitated after theaddition of beads. For the agitation, a conventional agitator may beadapted, or an apparatus suitable for the simultaneous processing ofmultiple specimens may be used. For magnetic separation, a conventionalmagnetic stand or a stand with an increased magnetic force adapted forthe processing of multiple specimens may be used.

Cancer determination is made by using the recovered cell-beads complex.For the determination of cancer, a material extracted from the cells orthe cells themselves are used. An automated system is constructed byadapting examination methods based on expression analysis utilizing aDNA chip or protein chip, or the identification of cancer cellsutilizing flow cytometry.

EXAMPLES

Hereafter, an example of the invention will be described. The invention,however, is not limited to the example.

(Recovery of Cells from Stool)

Stool voided by a colorectal cancer patient prior to operation was usedas a specimen. As to the use of stool, the subject was informed of thecontent of the procedure prior to the experiment and a written consentwas obtained.

Two hundred mL of Hanks solution (Nissui) containing 10% FBS was putinto a stomacher bag containing stool (about 5 to 80 g) and the bag wasthen sealed. A stool suspension was then prepared by using a stomacher(200 rpm, 1 min).

In the case where a stomacher bag having a filter was used, thesuspension was filtered by the filter in the bag. In the case where astomacher bag without a filter was used, the suspension was filtered bypassing it through a funnel-shaped filter set on a tubular plasticcontainer. The filtered solution was recovered in a beaker. The filteredsolution was further dispensed into five 50-mL centrifugal sedimentationtubes.

Forty μL of Ber-EP4 antibody-binding magnetic beads (DynabeadsEpithelial Enrich, Dynal Biotech) was added per centrifugalsedimentation tube, and the mixture was blended using a mix rotor(VMR-5, available from AS ONE) (at 4° C., 60 rpm, 30 min) so that thecells in the filtered solution were bound to the Ber-EP4 antibody.

After the individual centrifugal sedimentation tubes were set on amagnetic stand (Dynal MPC-1, Dynal Biotech), the stand was laid on amild mixer (SI-36, TAITEC Corporation) horizontally. The tubes were thensubjected to a seesaw motion for 15 minutes (60 reciprocations per min)to blend the filtered solution such that the magnetic beads werecollected on the side walls of the centrifugal sedimentation tubes.

After the filtered solution was removed, the centrifugal sedimentationtubes were detached from the stand, and 500 μL of Hanks solutioncontaining 10% FBS was added per tube to wash the beads collected on thewall surface.

The washing solution containing the beads was recovered into fiveEppendorf tubes (1.5 mL each) in which 500 μL of Hanks solutioncontaining 10% FBS had been put in advance. After a light suspension,the tubes were set on a magnetic stand (Dynal MPC-S, Dynal Biotech), andthe magnetic beads were collected on the side walls of the Eppendorftubes.

After the removal of the washing solution, the Eppendorf tubes weredetached from the stand, and 1 mL of Hanks solution containing 10% FBSwas added to each tube, and then the beads collected on the wall surfacewere washed. Similarly, the tubes were set on the magnetic stand and themagnetic beads were collected on the side walls of the Eppendorf tubes.The supernatant was then removed to obtain pellets of cell-beadscomplex. The recovery was conducted at room temperature.

(Cytological Analysis of the Recovered Cells)

The cell-beads complex pellets in each tube recovered in Example 1 weresuspended with the addition of 100 μL of YM fixing solution. Thesuspension was then transferred to a 50-mL centrifugal sedimentationtube. The total amount was adjusted to 25 mL by YM fixing solution,thereby obtaining a cell-containing fixing solution. The cell-containingfixing solution was then dispensed into an automatic smearing apparatusfor 8 glass slides. The apparatus was further filled with the YM fixingsolution and was then centrifuged at 2000 rpm for 10 minutes, therebysmearing the glass slides with the cells. After drying the slides withcold air, the cells were fixed with 95% ethanol.

The cells were then stained by the Papanicolaou staining method, whichis a representative method of staining for the observation of cellmorphology. The presence or absence of cancer cells were microscopicallyobserved and determined. The results are shown in Table 1.

Dukes No. Tumor site¹⁾ classification Cytodiagnosis Remarks²⁾ 1 S C + 2Ra B + 3 S A − Diarrhea due to Niflec 4 Rb C − Diarrhea due to Niflec 5Rb D − Clogged filter 6 S A − Clogged filter 7 Ra C + 8 Rb A + 9 Rb A +10 Ra A + 11 Rs A + 12 Ra A + 13 Rb A + 14 A A + 15 S C + 16 T A + ¹⁾S:Sigmoid colon Ra: Upper rectum Rb: Lower rectum Rs: S-shaped portion ofrectum A: Ascending colon T: Transverse colon ²⁾Nos. 1-12: Stomacherwith a filter was used. Nos. 13-16: Funnel-shaped filter was used.

Table 1 shows the cases of colorectal cancer patients who provided thespecimens used in the present experiment. Cytodiagnosis (+) indicatesthe cases where cancer cells were collected by the method of theinvention, and cytodiagnosis (−) indicates the cases where no recoveryof cancer cells were confirmed by the inventive method.

In cases Nos. 3 and 4, Niflec, a laxative, had been taken by the patientprior to the passage of stool, so that the voided stool was diarrhealfrom which cells could not be recovered. In cases Nos. 5 and 6 too, nocells could be recovered. In these two cases, the amount of stoolexceeded 100 g and, in addition, a stomacher bag with a filter was usedat this point for filtering the stool suspension, resulting in asignificant clogging of the filter. The reduction in the cell collectionrate depending on the extent of clogging of the filter had also beenanticipated in a preliminary experiment, and it was clearly shown thatan excessive clogging of the filter would prevent the recovery of cells.

Thus, cells were recovered from 12 out of 16 cases (75%). Even in caseswhere no cell could be recovered, the reasons for the absence ofrecovery were obvious, as mentioned above. Thus, it was clearly shownthat cells can be very efficiently recovered from colorectal cancerpatients by using the magnetic beads method in accordance with thepresent protocol.

When the progress of cancer in the colorectal cancer patients whoprovided the specimens was examined, 8 out of the 12 cases from whichcells were recovered (67%) were classified into early cancer of Dukes A.In addition, cells could be recovered from cases of Dukes A involvingthe tumor sites of upper rectum (No. 14) or transverse colon (No. 16).These results show that the method of the invention is very effective indiagnosing early cancer including those cancers developed at sites wheredetection by colonoscopy, for example, is difficult.

(Analysis of the Conditions for the Magnetic-Beads Cell Recovery MethodUsing Cultured Cells)

A colorectal cancer cultured cell line (HT-29) was mixed in a suspensionof stool collected from an infant, and the mixture was reacted with aBer-EP4 antibody-binding magnetic bead. The conditions that affect thecell recovery rate in the present method were then examined. Thesuspension of the stool from infant was filtered by the above-describedfunnel-shaped filter to obtain a filtered solution. The recovery ratewas calculated by measuring the number of cells that bound to therecovered beads using Nucleo Counter (from M&S TechnoSystems) andcomparing it with the number of cells initially added to the suspension.

First, the temperature suitable for the binding of beads and cells wasexamined. Magnetic beads and cells are in many cases reacted at 4° C.This is for the purpose of lowering the damage done to the cells orpreventing the phenomena in which macrophages contained in the specimenprey on the beads, for example.

Thus, a mixing reaction between a 25-mL cell−(8.4×10⁵) stool suspensionand 40 μL of beads was conducted at 4° C. and at room temperature. As aresult, it was revealed that the same level of cell recovery rate can beobtained at room temperature as at 4° C. (FIG. 5A). This indicates thatall of the cell recovery steps of the method of the invention can beconducted at room temperature, whereby individual operations can begreatly simplified as compared with the aforementioned cooling method.

Next, the need for the blood serum in the cultured solution forsuspension was analyzed. Blood serum was expected to have functions forimproving the efficiency of the magnetic beads method by, for example,restricting the protease activity in the solution and stabilizing thecells, or preventing the adsorption of non-specific cells.

Accordingly, 25 mL of cell-stool suspension was prepared using a Hankssolution containing 10% blood serum (FBS, fetal bovine serum) and aHanks solution containing no blood serum, and the suspension was reactedwith 40 μL of beads, in order to examine the cell recovery rate in asimilar manner.

The results (FIG. 5B) showed that the cell recovery rate was lower inthe case where no blood serum was contained, thus indicating theeffectiveness of blood serum in the recovery method of the invention.

Further, the size of the mesh of the funnel-shaped filter used in thepresent experiment was analyzed. Nylon filters of different sizes (1000,512, 96 and 48 μm), with the entire surface made of mesh and a shape asshown in FIG. 3, were prepared. Cultured cells were added to a stoolsuspension in a similar manner, filtered by these four kinds of filters,and then reacted with beads to examine the cell recovery rate.

As a result, the recovery rate of the filter of 96 μm was about one halfof that of 512 μm, and the rate for 48 μm was about one fifth of thatfor 512 μm. The filter of 1000 μm had approximately the same recoveryrate as that of the filter of 512 μm. These results thus indicated thatthe size of filter should preferably be 500 or more. However, in orderfor the filter to function as such, the size should not exceed 1500 μm.

(Comparison with the Percoll Centrifugation Method)

The cell recovery rate of the magnetic beads method was compared withthat of the Percoll centrifugation method, which is a conventionalmethod. The experiment was conducted using cultured cells in a mannersimilar to those described above. The Percoll centrifugation method wascarried out in accordance with the method of Yamao et al. reported inNon-Patent Document 4. In the Percoll centrifugation method, Percollsolution is mixed with cells and is then centrifuged in order toseparate the cells according to their densities. The results are shownin FIG. 6. The cell recovery rate was 0.8% for the Percollcentrifugation method. The recovery rate for the magnetic beads method(in accordance with the standard cell recovery protocol was 66.7%, thusindicating the advantage of the magnetic beads method.

1. A method of recovering a cancerous cell comprising: a step ofpreparing a sample which comprises adding a buffer solution to acollected naturally voided stool at 15-35° C., wherein the amount ofsaid buffer solution is equal to or more than the amount of said stool;a step of removing an impurity from said sample by using a filter; astep of adsorbing cancerous cell sample, from which an impurity isremoved, on a magnetic bead on the surface of which is immobilized anantibody against an antigen on the surface of an epithelial cell and/orepithelial cancer cell at 15-35° C.; and a step of recovering the thusadsorbed cancer cell at 15-35° C. by using a magnet.
 2. The method ofrecovering a cancerous cell according to claim 1, wherein said buffersolution contains blood serum.